Edelfosine Induces an Apoptotic Process in Leishmania infantum That Is Regulated by the Ectopic Expression of Bcl-X_L and Hrk

Abstract

The alkyl-lysophospholipids edelfosine and miltefosine induce apoptosis in Leishmania infantum promastigotes. The finding that edelfosine-induced cell death can be regulated by the ectopic expression of the antiapoptotic and proapoptotic members of the Bcl-2 family of proteins Bcl-X_L and Hrk suggests that this process is similar to apoptosis in eukaryotic cells.

Miltefosine, edelfosine, and several other alkyl-lysophospholipids originally developed as anticancer agents have proved to be effective antileishmanial drugs (4, 7). An increasing number of reports have shown that culture saturation (3), heat shock treatment (1, 5), and exposure to chemotherapeutic agents such as glucantime (8) and miltefosine (6, 9) induce features of programmed cell death (PCD) on Leishmania parasites. To gain further insight into this process, we have investigated whether proteins that regulate apoptosis in higher eukaryotes have any effect on edelfosine-induced PCD in Leishmania infantum.

Exponentially growing (2 × 10⁶ parasites/ml) L. infantum (M/CAN/ES/96/BCN150 MON-1) promastigotes were subjected to incubation with increasing concentrations of either edelfosine or miltefosine for 24 h, and the percentage of dead parasites was evaluated by flow cytometry after the parasites were stained with 5 μM propidium iodide (PI) (1). The increase in drug concentrations correlated with the percentages of PI staining-positive parasites, which was an indication of the cytotoxic effects of the drugs (Fig. 1A and B). The estimated 50% lethal doses were 27 μM (R² > 0.99) for edelfosine and 47 μM (R² > 0.99) for miltefosine. The DNA content in the drug-treated parasites was analyzed by flow cytometry (1). Both drugs induced a concentration-dependent process of DNA degradation, as shown by the progressive increase in the percentages of hypodiploid cells (Fig. 1C and D). The results also reveal that edelfosine has greater potency than miltefosine against L. infantum promastigotes.

FIG. 1.

Leishmanicidal effects of edelfosine and miltefosine. (A and B) Percentages of PI-positive L. infantum promastigotes after 24 h of treatment with increasing concentrations of edelfosine (A) or miltefosine (B); (C and D) percentages of hypodiploid promastigotes after 24 h of treatment with increasing concentrations of edelfosine (C) or miltefosine (D); (E and F) monoparametric histograms comparing the relative TMRM-derived fluorescence of control and 45 μM edelfosine-treated (E) or 45 μM miltefosine-treated (F) promastigotes. LD₅₀, 50% lethal dose.

Changes in the mitochondrial transmembrane potential (ΔΨ_m) were analyzed by flow cytometry after staining of the parasites with tetramethylrhodamine methyl ester (TMRM) (2). As already shown for heat-induced cell death (1), edelfosine or miltefosine treatment causes a nonhomogeneous effect on the ΔΨ_m of the parasites (Fig. 1E and F). After 24 h, the parasites can be divided into two populations according to their mitochondrial status: the first one is composed of parasites with a reduced ΔΨ_m, and the second one is composed of a population in which the parasites show a clear increase in ΔΨ_m compared to that for the untreated controls. This increase in ΔΨ_m can be observed as soon as 30 min after drug treatment (data not shown) and may be interpreted as a strategy that the cell uses to obtain enough energy to develop the apoptotic process. The observed decrease in ΔΨ_m, together with the presence of a sub-G₁ peak in the cell cycle analysis, is suggestive of a death process similar to that of apoptosis in response to edelfosine or miltefosine. The induction of apoptosis following miltefosine treatment has already been reported in Leishmania donovani (6, 9, 10).

Apoptosis in higher eukaryotes is regulated by members of the Bcl-2 family of proteins (11). When parasites transfected with a pX63-Neo vector containing the bcl-X_L-coding sequence were treated with edelfosine, significant decreases in the number of hypodiploid cells (Fig. 2A) and the number of cells present in the population with a low ΔΨ_m (Fig. 2B) were observed. Our results clearly indicate that both apoptotic processes are partially reverted by Bcl-X_L expression.

FIG. 2.

Proteins from the Bcl-2 family modulate edelfosine-induced cell death in L. infantum promastigotes. Transfected L. infantum strains carrying the empty vector (pX63) or the genes encoding the antiapoptotic Bcl-X_L (BclXL) or proapoptotic Hrk were exposed to 40 μM edelfosine for 24 h. Cell death was measured in terms of DNA degradation (A) and the decrease in the mitochondrial membrane potential (B). Light gray bars, untreated parasites; black bars, edelfosine (Edf)-treated parasites; lines above the bars, standard deviations (n = 3); *, P < 0.05. (C) Confocal microscopy images of L. infantum strains transfected with empty pX63 (L. infantum), pX63-bcl-X_L (L. infantum-Bcl-XL), or pX63-hrk (L. infantum-Hrk) and incubated in the absence of edelfosine (control) or exposed to 40 μM edelfosine for 24 h and then analyzed by the TUNEL assay, PI staining, and differential interference contrast (DIC). (D) Western blot analysis of the Leishmania strains expressing either Bcl-X_L or HrK. Detection of the ectopic proteins expressed was carried out with anti-HA (Bcl-X_L) or anti-FLAG (Hrk) antibodies. Antibodies against the Kmp-11 protein were used to confirm that the same amount of protein was loaded in each lane.

To check whether a proapoptotic member of the Bcl-2 family could also modify the response of the cell population to edelfosine, L. infantum promastigotes were transfected with a pX63-Neo vector containing the hrk-coding sequence. Significant increases in the percentage of hypodiploid parasites (Fig. 2A) and the number of promastigotes with low ΔΨ_m values (Fig. 2B) were observed when these transfected promastigotes were treated with 40 μM edelfosine for 24 h.

The effect of Bcl-X_L or Hrk expression was further confirmed by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick-end-labeling (TUNEL) method (Fig. 2C). Statistical analysis of the images (Table 1) revealed that the percentages of fluorescent cells in nontransfected parasites and in parasites transfected with an empty vector (pX63) were similar (42.5% and 47.4%, respectively), whereas only 26.7% of the parasites expressing the antiapoptotic Bcl-X_L was TUNEL assay positive. On the other hand, 92.5% of the parasites expressing the proapoptotic Hrk were positive for staining by the TUNEL assay, which confirms the proapoptotic effect of the expression of this protein in Leishmania parasites.

TABLE 1.

Measurement of apoptosis by TUNEL technique^a

Cells	Treatment	% TUNEL assay-positive cells
L. infantum wild type	Control	0
L. infantum wild type	40 μM edelfosine, 24 h	42.5 ± 4.7
L. infantum pX63	Control	0
L. infantum pX63	40 μM edelfosine, 24 h	47.4 ± 5.2
L. infantum Bcl-XL	Control	0
L. infantum Bcl-XL	40 μM edelfosine, 24 h	26.7 ± 3.1
L. infantum Hrk	Control	0
L. infantum Hrk	40 μM edelfosine, 24 h	92.5 ± 8.3

^aApoptosis was measured by the TUNEL technique with untreated control cells and cells treated with 40 μM edelfosine for 24 h. Wild-type parasites and parasites transfected with the empty expression vector pX63 (L. infantum pX63), pX63-bcl-X_L (L. infantum Bcl-X_L), or pX63-hrk (L. infantum Hrk) were used. The data are shown as the means ± standard deviations (n = 3).

We have previously shown that Bcl-X_L is able to partially revert heat shock-induced cell death in L. infantum promastigotes (1). The prosurvival activity of this protein was also confirmed in the present work. On the other hand, the expression of Hrk severely impairs the ability of L. infantum promastigotes to survive in the presence of edelfosine. Conservation of the antiapoptotic and proapoptotic activities of Bcl-X_L and Hrk, respectively, in Leishmania promastigotes may be considered an indication of the presence of BH3-bearing proteins, which have been implicated in the regulation of cell death, in the Leishmania proteome.